The centrosome is of fundamental importance to animal cells in that it is the principal nucleator of the microtubule (MT) cytoskeleton and harbors the centrioles, specialized structures that are required for cilia formation and contribute to spindle-pole positioning. Through these principal roles, the centrosome is important in embryonic development and human disease. During interphase, MTs are required for vesicle trafficking and cell polarity. In most cell types, many of the MTs that form the mitotic spindle originate at the centrosome. The centrosome also has important roles beyond MT nucleation, including functions in cytokinesis, progression through the cell cycle, and in sequestering or positioning some proteins to control when and where they are active. The primary aims of this proposal are to determine the molecular mechanism of MT nucleation, and to understand how the nucleating machinery is assembled and regulated. Moreover we will begin to explore the mechanism by which microtubule doublets and triplets are formed at the centriole by focusing on the newly discovered ?- and ?-tubulins and their as yet unknown, binding partners. Our laboratory is uniquely poised to utilize a hierarchical array of structural approaches (x-ray crystallography, electron microscopic (EM) single particle reconstruction, and EM Tomography, small-angle x-ray scattering (SAXS)) to determine the structures of ?-, ?- and ?-tubulin complexes in vitro and in situ, and to understand their mechanism of action through functional studies and innovative kinetic modeling. By combining structural studies, MT assembly experiments and kinetic modeling, our previous work is leading to a paradigm shift in understanding the underlying principles of MT formation, suggesting a new role for GTP, a different model for MT assembly and an unexpected mode of regulation of nucleation in the ?-tubulin complexes. The proposed experiments will continue and expand upon these results, providing a detailed understanding of the physical origins of MT assembly, and the cellular machinery that dictates MT formation. In vitro predictions of modes of action or regulation will be assayed in vivo using mutagenesis and siRNA and live imaging. Spanning size scales from the atomic to the entire organelle, our long-term goal is to synthesize an atomic resolution picture of all the relevant structural and functional interactions between 12-tubulin, ?-, ?- and ?-tubulin complexes, regulatory proteins and the centrosomal matrix. Public Health Relevance: The normal function of centrosomes and centrioles is directly relevant to human disease, and the proposed work is aimed at understanding the molecular mechanisms of these cellular components. Abnormal centrosome number and behavior is a common hallmark of cancer cells, and centriole defects are involved in many human ciliary diseases, including nephronophthisis, Bardet-Biedl syndrome, Meckel syndrome, and Oral-Facial-Digital syndrome.